The long-term goal of these experiments is to resolve the structural and functional organization of the neural networks responsible for contrOlling neuroendocrine CRH neurons in the paraventricular nucleus of the hypothalamus (PVH). The PVH is the key convergence point for the neural regulation of the hypothalamo-pituitary-adrenal axis. The proposed studies will focus on interactions between catecholaminergic inputs to the PVH and neuroendocrine CRH neurons. Catecholaminergic afferents are one of the densest and most widely studied inputs to the PVH. They playa critical role in generating ACTH and glucocorticoid responses to a wide range of stressors, but particularly those generated within the body such as cardiovascular and metabolic stimuli. During psychological stress, these inputs also help the PVH coordinate neuroendocrine and autonomic responses with the broader responses generated by the telencephalon. The proposed experiments use a hierarchically-ordered model of the afferent inputs to CRH neurons, which generates a number of testable hypotheses. One of these is the focus of this proposal: neuropeptide Y, which is colocalized in many catecholaminergic afferents, has major stimulatory effects on CRH neurons and ACTH release, and potentiates the effects of catecholamines on CRH neurons. Experiments will use three complementary approaches. First, the effects of selectively removing the CAergic input to the PVH on the ability of PVH-directed injections of NPY to activate CRH neurons will be determined. Second, in vivo experiments will determine the ability of various NPY antagonists to block CRH neuronal responses to intra-venous 2-deoxy-d-glucose (2DG) injections. Third, acutely-prepared ex vivo hypothalamic slices will be used to investigate how NPY and norepinephrine interact to control CRH neuronal responses. EndpOints will focus on CRH synthesis and release, and will include CRH hnRNA, phospho-ERK1/2, phospho-CREB, and ACTH release. Analytical methods include in situ hybridization, fluorescence immunocytochemistry detected with confocal and conventional microscopy, radioimmunoassay, and RT -qPCR. Collectively, the experiments in this project are designed to make major contributions towards elucidating the organization and function of the. neural networkS responsible for controlling CRH neuroendocrine function: